Triazole antifungal agents

ABSTRACT

An antifungal compound of formula (I):                    
     or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, wherein X is CH or N; R 1  is phenyl subsituted with 1 to 3 substituents each independently selected from halo and CF 3 ; R 2  is (hydroxy)C 1 -C 4  alkyl, CONH 2 , S(O) m (C 1 -C 4  alkyl), Ar or Het; m is 1 or 2; Ar is phenyl optionally monosubstituted with halo or CF 3 ; and Het is a C-linked 6-membered nitrogen-containing aromatic heterocyclic group containing 1 or 2 nitrogen atoms, or a C- or N-linked 5-membered nitrogen-containing aromatic heterocyclic group containing from 2 to 4 nitrogen atoms, wherein either of said heterocyclic groups is optionally substituted with C 1 -C 4  alkyl or (C 1 -C 4  alkoxy)methyl.

This application is a divisional of application Ser. No. 09/365,929,filed on Aug. 2, 1999 and now U.S. Pat. No. 6,124,325, which is adivisional of application Ser. No. 08/828,754, filed Mar. 18, 1997 andnow U.S. Pat. No. 5,981,560.

This invention relates to triazole derivatives which have antifungalactivity and are useful in the treatment of fungal infections inanimals, including human beings.

Thus the invention provides compounds of formula (I):

pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity, wherein

X is CH or N;

R¹ is phenyl subsituted with 1 to 3 substituents each independentlyselected from halo and CF₃;

R² is (hydroxy)C₁-C₄ alkyl, CONH₂, S(O)_(m)(C₁-C₄ alkyl), Ar or Het; mis 1 or 2;

Ar is phenyl optionally monosubstituted with halo or CF₃; and

Het is a C-linked 6-membered nitrogen-containing aromatic heterocyclicgroup containing 1 or 2 nitrogen atoms, or a C- or N-linked 5-memberednitrogen-containing aromatic heterocyclic group containing from 2 to 4nitrogen atoms, wherein either of said heterocyclic groups is optionallysubstituted with C₁-C₄ alkyl or (C₁-C₄ alkoxy)methyl.

In the above definition, unless otherwise indicated, alkyl and alkoxygroups having three or more carbon atoms may be straight or branchedchain; halo means fluoro, chloro, bromo or iodo. In addition. Het isselected from pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl,imidazolyl, triazolyl or tetrazolyl.

The compounds of formula (I) contain at least two chiral centres andtherefore can exist as stereoisomers, i.e. as enantiomers ordiastereoisomers, as well as mixtures thereof. The invention includesboth the individual stereoisomers of the compounds of formula (I)together with mixtures thereof. Separation of diastereoisomers may beachieved by conventional techniques, e.g. by fractional crystallisationor chromatography (including HPLC) of a diastereoisomeric mixture of acompound of formula (I) or a suitable salt or derivative thereof. Anindividual enantiomer of a compound of formula (I) may also be preparedfrom a corresponding optically pure intermediate or by resolution,either by HPLC of the racemate using a suitable chiral support or byfractional crystallisation of the diastereoisomeric salts formed byreaction of the racemate with a suitable optically active acid.

The preferred stereoisomers of formula (I) have the(2R,3S)-configuration of formula (IA):

Furthermore, the compounds of formula (I) may exist as cis- ortrans-alkene isomers and the invention also includes both separateindividual isomers and mixtures thereof. The preferred isomers are thetrans-isomers.

Certain compound of formula (I) may also exist in tautomeric forms andthe invention includes both separate individual tautomers and mixturesthereof.

Also included in the invention are radiolabelled derivatives ofcompounds of formula (I) which are suitable for biological studies.

The pharmaceutically acceptable salts of the compounds of formula (I)are, for example, non-toxic acid addition salts formed with inorganicacids such as hydrochloric, hydrobromic, sulphuric and phosphoric acid,with organo-carboxylic acids, or with organo-sulphonic acids. Certaincompounds of formula (I) can also provide pharmaceutically acceptablemetal salts, in particular non-toxic alkali metal salts, with bases.Examples include the sodium and potassium salts. For a review ofsuitable pharmaceutical salts, see J. Pharm. Sci., 1977, 66, 1.

A preferred group of compounds of formula (I) is that wherein R¹ isphenyl substituted by 1 or 2 substituents each independently selectedfrom F and Cl; R² is hydroxypropyl, CONH₂, SO₂CH₃, Ar or Het; Ar isfluorophenyl; Het is a pyridyl, pyrazolyl, imidazolyl or triazolylgroup, wherein, said pyrazolyl group is substituted with methyl and saidtriazolyl group is optionally substituted with ethoxymethyl; and X is aspreviously defined for formula (I).

A more preferred group of compounds of formula (I) is that wherein R¹ is2,4-difluorophenyl; R² is C(CH₃)₂OH, CONH₂, SO₂CH₃, 4-fluorophenyl,2-pyridyl, 1-methylpyrazol-5-yl, imidazol-1-yl, 1,2,3-triazol-4-yl or1-ethoxymethyl-1,2,3-triazol-5-yl; and X is as previously defined forformula (I).

Particularly preferred compounds of the invention include:

trans-(2R,3S)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[2-(1-methylpyrazol-5-yl)ethenyl]phenyl}butan-2-ol;

trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[2-(imidazol-1-yl)ethenyl]phenyl}butan-2-ol;and

trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[5-(2-carbamoylethenyl)pyrid-2-yl]butan-2-ol;

and pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity.

In another aspect, the present invention provides processes for thepreparation of compounds of formula (I), their pharmaceuticallyacceptable salts, and pharmaceutically acceptable solvates of eitherentity.

A compound of formula (I) may be prepared from a compound of formula(II):

wherein Z is bromo or iodo, and X and R¹ are as previously defined forformula (I), by treatment with a compound of formula (III):

CH₂═CHR²  (III)

wherein R² is as previously defined for formula (I), under typical Heckreaction conditions. The reaction is generally carried out using fromabout a 20 to about a 100% excess of the required alkene and from abouta 50 to about a 100% excess of a tertiary amine, in the presence of fromabout 0.05 to about 0.60 equivalent of a palladium salt and from about0.10 to about 1.10 equivalents of a tertiary arylphosphine, in asuitable solvent such as acetonitrile or dimethylformamide, at fromabout 80 to about 160° C. Preferably the tertiary amine istriethylamine, the palladium salt is palladium acetate, the phosphine iseither tri-o-tolylphosphine or 1,1′-bis(diphenylphosphino)ferrocene, andthe reaction is conducted in refluxing acetonitrile.

A compound of formula (II) may be prepared by a variety of syntheticprocedures. For example one such procedure, which is preferred when X isN, involves the reaction of a compound of formula (IV):

wherein R¹ is as previously defined for formula (II), with anorganometallic compound of formula (V):

wherein M is a suitable metal (e.g. lithium, sodium or potassium) ormetal halide (e.g. magnesium halide or zinc halide), and X and Z are aspreviously defined for formula (II).

An organometallic compound of formula (V) wherein M is a suitable metalis preferably generated in situ by deprotonation of the correspondingalkane precursor (i.e. a compound of formula (V) wherein M is hydrogen)with a suitable base, e.g. lithium or potassium diisopropylamide orlithium, sodium or potassium bis(trimethylsilyl)amide.

An organometallic compound of formula (V) wherein M is a suitable metalhalide, e.g. a Grignard reagent or organozincate, can be prepared eitherby treatment in situ of the corresponding organometallic compound offormula (V) wherein M is lithium with a suitable metal halide, e.g.magnesium bromide or zinc iodide, or by treatment of the correspondingalkyl halide precursor (i.e. a compound of formula (V) wherein M ischloro, bromo or iodo) with magnesium or zinc respectively, optionallyusing iodine to promote the reaction.

Preferably (V) wherein M is chloro, bromo or iodo is converted to thecorresponding zincate in the presence of (IV) in a suitable solvent atabout room temperature in an inert atmosphere by treating it with zincin the presence of iodine. This may be achieved in tetrahydrofuran assolvent using about 2.6 equivalents of zinc powder, followed by 0.2equivalent of iodine, which leads to an exothermic reaction.

The compounds of formula (IV) are either known, e.g. see EP-A-044605,EP-A-069442 or GB-A-1464224, or may be prepared by methods similar tothose described therein.

An alternative synthetic procedure for preparing a compound of formula(II), which is preferred when X is CH, involves the reduction of acompound of formula (VI):

wherein Z, X and R¹ are as previously defined for formula (II).

The reduction is conveniently effected using diimide generated in situ.Thus diimide precursor, such as p-toluenesulphonylhydrazide, and (VI)are combined in a suitable solvent, e.g. toluene, and the reactionconducted at the reflux temperature of the reaction medium.

The reduction may also be carried out by catalytic hydrogenation using asuitable catalyst such as palladium on charcoal in an appropriatesolvent, e.g. a C₁-C₃ alkanol.

A compound of formula (VI) may be prepared by reaction of an epoxide offormula (VII):

wherein Z, X and R¹ are as previously defined for formula (VI), with1,2,4-triazole in the presence of a base or with a tetraalkylammonium oralkali metal salt (preferably the sodium salt) of 1,2,4-triazole in asuitable solvent such as dimethylformamide, methanol or aqueous acetone.The reaction is conveniently carried out using about a 50% excess of thesaid sodium salt in dry dimethylformamide at about 70° C.

The resulting racemic mixture of 2R and 2S enantiomers may beconveniently resolved at this stage, e.g. by chromatography using achiral stationary phase, and the 2R enantiomer reduced as above toafford, after further chromatographic resolution, the preferred 2R, 3Senantiomer of a compound of formula (II).

A compound of formula (VII) may be prepared by methylenation of a ketoneof formula (VIII):

wherein Z, X and R¹ are as previously defined for formula (VII), understandard Wittig or Wittig-Horner reaction conditions. For example,(VIII) is treated with about a 30% excess of the ylid generated in situfrom methyltriphenyl-phosphonium bromide and a strong base, e.g.n-butyllithium in hexane solution, in a suitable solvent such as drytetrahydrofuran at from about −20° C. to about room temperature in aninert atmosphere.

A compound of formula (VIII) may be prepared by epoxidation of an alkeneof formula (IX):

wherein Z, X and R¹ are as previously defined for formula (VIII).

Of the plethora of oxidation reagents and reactions available, aconvenient technique is the use of phase transfer catalysis using, forexample, a quatenary ammonium salt as catalyst. Typical conditions areto employ about a 10% excess of an oxidant such as t-butyl hydroperoxidein a suitable solvent, e.g. toluene, in the presence of about 0.1equivalent of benzyltrimethylammonium hydroxide in aqueous solution atabout room temperature.

A compound of formula (IX) may be prepared by methylenation of acompound of formula (X):

wherein Z, X and R¹ are as previously defined for formula (IX), using aMannich-type reaction. This may be conveniently achieved by treating (X)in the presence of about a 5-fold excess of acetic anhydride with abouta 50% excess of bis(dimethylamino)methane at about room temperature.

A compound of formula (X) may be prepared by any of a myriad of standardα-methyleneketone syntheses. For example, a substituted benzyl halide offormula (Xl):

R¹CH₂Y  (XI)

wherein Y is chloro, bromo or iodo and R¹ is as previously defined forformula (X), is converted to the corresponding Grignard reagent which isthen reacted with about a 40% excess of a hydroxamic acid derivative offormula (XII):

wherein Z and X are as previously defined for formula (X), in a suitablesolvent such as dry ether at from about −70° C. to about roomtemperature in an inert atmosphere.

Certain compounds of formula (I) wherein R² is a C-linked 5-memberednitrogen-containing aromatic heterocyclic group containing from 2 to 4nitrogen atoms substituted on a nitrogen atom with C₁-C₄ alkyl or (C₁-C₄alkoxy)methyl can be prepared by N-alkylation of the correspondingunsubstituted compounds, e.g. by using the appropriate C₁-C₄ alkylhalide or (C₁-C₄ alkoxy)methyl halide (e.g. chloride or bromide),typically in the presence of an acid acceptor in a suitable solvent.When tautomerism of the ring is possible, alkylation may occur on one ormore nitrogen atoms but the resulting mixture of products can beseparated by chromatograpy.

Alternatively, the reverse transformation may be exploited. For example,a compound of formula (I) bearing a N-(C₁-C₄ alkoxy)methyl substituentwithin R² may be converted to the corresponding unsubstituted derivativeby acid hydrolysis in a suitable solvent. The reaction can beconveniently carried out using dilute hydrochloric acid in aqueousethanol as solvent at the reflux temperature of the reaction medium.

A compound of formula (III) may be prepared by methylenation of thecorresponding aldehyde (R²CH═O) by Wittig or Wittig-Horner methodologyas described for the conversion of (VIII) to (VII). Where necessary, therequired aldehydes are obtained by formylation, e.g. usingdimethylformamide, of the corresponding (hetero)aryllithium understandard reaction conditions.

The intermediate alkane and alkyl halide precursors of (V) andintermediates, of formulae (III), (XI) and (XII), when neithercommercially available nor subsequently described, can be obtainedeither by analogy with the processes described in the Preparationssection or by conventional synthetic procedures, in accordance withstandard textbooks on organic chemistry or literature precedent, fromreadily accessible starting materals using appropriate reagents andreaction conditions.

Moreover, persons skilled in the art will be aware of variations of, andalternatives to, those processes described hereinafter in the Examplesand Preparations sections which allow the compounds defined by formula(I) to be obtained.

The pharmaceutically acceptable acid addition salts of the compounds offormula (I) may also be prepared in a conventional manner. For example asolution of the free base is treated with the appropriate acid, eitherneat or in a suitable solvent, and the resulting salt isolated either byfiltration or by evaporation under reduced pressure of the reactionsolvent. Pharmaceutically acceptable base addition salts can be obtainedin an Analogous manner by treating a solution of a compound of formula(I) with the appropriate base. Both types of salt may be formed orinterconverted using ion-exchange resin techniques.

The compounds of formula (I), their pharmaceutically acceptable salts,and pharmaceutically acceptable solvates of either entity are antifungalagents, useful in the curative or prophylactic treatment of fungalinfections in animals, including human beings. For example, they areuseful in treating superficial fungal infections in humans caused by,among other organisms, species of Candida, Trichophyton, Microsporum orEpidermophyton, or in mucosal infections caused by Candida albicans(e.g. thrush and vaginal candidiasis) and they can also be used in thetreatment of systemic fungal infections caused by, for example, speciesof Candida (e.g. Candida albicans), Cryptococcus neoformans, Aspergillusflavus, Aspergillus fumigatus, Coccidioides, Paracoccidioides,Histoplasma or Blastomyces. Indeed, they possess potent, broad spectrumantifungal activity both in vitro and in vivo.

Certain compounds of the invention have been found to have unexpectedlygood broad spectrum activity, including excellent activity against theclinically important Aspergillus spp. fungi.

The in vitro evaluation of the antifungal activity of the compounds canbe performed by determining the minimum inhibitory concentration(m.i.c.), which is the concentration of a test compound, in a suitablemedium, at which growth of the particular micro-organism fails to occur.In practice, a series of agar plates, or liquid medium in microtiterplates, each having the test compound incorporated at a particularconcentration, is inoculated with a standard culture of, for example,Cryptococcus neoformans, and each plate is then incubated for 48 hoursat 37° C. The plates are then examined for the presence or absence ofgrowth of the fungus and the appropriate m.i.c. value is noted. Othermicro-organisms used in such tests can include Candida albicans,Aspercillus fumigatus, Trichophyton spp., Microsporum spp.,Epidermophyton floccosum, Coccidioides immitis and Torulopsis glabrata.

The in vivo evaluation of the compounds can be carried out at a seriesof dose levels by intraperitoneal or intravenous injection, or by oraladministration, to mice or rats which are inoculated with, e.g. a strainof Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans.Activity may be based on the number of survivors from a treated group ofmice after the death of an untreated group of mice.

For Candida spp. infection models the dose level at which the compoundprovides 50% protection against the lethal effect of the infection(PD₅₀) is also assessed.

For Aspergillus spp. infection models the number of mice cured of theinfection after a set dose allows further assessment of activity.

For Cryptococcus spp. infection models the number of colony formingunits existing after a set dose is assessed and compared with control todetermine compound efficacy. A preliminary assessment of potential livertoxicity may also be made on the basis of increase in liver weightrelative to control.

For human use, the antifungal compounds of the invention can beadministered alone, but will generally be administered in admixture witha pharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. For example, theycan be administered orally in the form of tablets containing suchexcipients as starch or lactose, or in capsules or ovules either aloneor in admixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents. They can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, they are best used in theform of a sterile aqueous solution which may contain other substances,for example enough salts or glucose to make the solution isotonic withblood.

The solubility of a compound of formula (I) in an aqueous medium may beimproved by complexation with a hydroxyalkyl (see EP-A-0149197) orsulfoalkyl (see WO 91/11172) derivative of a cyclodextrin in thepreparation of an appropriate pharmaceutical composition. Preferably thecyclodextrin used is alpha-, beta- or gamma-cyclodextrin.

For oral and parenteral administration to human patients, the dailydosage level of the antifungal compounds of the invention will be from0.01 to 20 mg/kg (in single or divided doses). Thus tablets or capsulesof the compounds will contain from 5 mg to 0.5 g of active compound foradministration singly, or two or more at a time, as appropriate. Thephysician in any event will determine the actual dosage which will bemost suitable for an individual patient and it will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case; there can, of course, be instances wherehigher or lower dosage ranges are merited and such are within the scopeof this invention.

Alternatively, the antifungal compounds of the invention can beadministered in the form of a suppository or pessary, or they may beapplied topically in the form of a lotion, solution, cream, ointment ordusting powder. For example, they can be incorporated into a creamconsisting of an aqueous emulsion of polyethylene glycols or liquidparaffin, or they can be incorporated, at a concentration of from 1 to10%, into an ointment consisting of a white wax or white soft paraffinbase together with such stabilisers and preservatives as may berequired.

Thus the invention provides a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable solvate of either entity, together witha pharmaceutically acceptable diluent or carrier.

The invention also provides a compound of formula (I), or apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for use as a medicament.

The invention further includes the use of a compound of formula (I), ora pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for the manufacture of a medicament forthe curative or prophylactic treatment of fungal infections.

In a further aspect, the invention provides a method of treating ananimal (including a human being) to cure or prevent a fungal infection,which comprises treating said animal with an effective amount of acompound of formula (I), or a pharmaceutically acceptable salt, or apharmaceutically acceptable solvate of either entity, or apharmaceutical composition containing any of the foregoing.

The invention also includes any novel intermediates described herein,e.g. the compounds of formula (II).

The syntheses of the compounds of the invention and of the intermediatesfor use therein are illustrated by the following Examples andPreparations.

¹H Nuclear magnetic resonance (NMR) spectra were recorded using either aNicolet QE-300 or a Bruker AC-300 spectrometer and were in all casesconsistent with the proposed structures. Characteristic chemical shifts(δ) are given in parts-per-million downfield from tetramethylsilaneusing conventional abbreviations for designation of significant peaks:e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.

Mass spectra (m/z) were obtained with a Fisons Instruments Trio 1000spectrometer using thermospray ionisation.

Room temperature means 20-25° C.

EXAMPLE 1Trans-(2R,3S)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[2-(1methylpyrazol-5-yl)ethenyl]phenyl}butan-2-ol

A stirred solution of(2R,3S)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4-iodophenyl)butan-2-ol(Preparation 12; 0.5 g, 1.1 mmol), 1-methyl-5-vinylpyrazole (Preparation17; 0.14 g, 1.3 mmol), triethylamine (0.25 ml, 2 mmol), palladiumacetate (130 mg, 0.6 mmol) and tri-o-tolylphosphine (340 mg, 1.2 mmol)in acetonitrile (25 ml) was heated under reflux for 1 hour and thenevaporated under reduced pressure. The residue was partitioned betweenethyl acetate (100 ml) and saturated aqueous sodium bicarbonate solution(50 ml), then the organic phase separated, washed with saturated brine(30 ml), dried (Na₂SO₄) and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel, using anelution gradient of hexane:propan-2-ol (90:10 to 75:25), to give thetitle compound (0.31 g) as a white solid, m.p. 81-83° C., aftercrystallisation from aqueous ethanol. [α]_(D) ²⁵ −55° (c=0.1, CH₃OH).

δ(CDCl₃): 1.10(3H,d), 3.30(1H,q), 3.90 and 4.80(2H, AB system),3.95(3H,s), 4.75(1H,s), 6.45(1H,d), 6.80(2H,m), 7.00(2H, AB system),7.50(6H,m), 7.70(2H,m). Found: C,64.77; H,5.19; N,15.65. C₂₄H₂₃F₂N₅O;0.50 H₂O requires C,64.85; H,5.44; N,15.76%. m/z 436 (M+1)⁺.

EXAMPLE 2Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-[2-(4-fluorophenyl)ethenyl]pyrid-2-yl}butan-2-ol

A stirred solution of(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(5-bromopyrid-2-yl)butan-2-ol(Preparation 3; 0.5 g, 1.22 mmol), 4-fluorostyrene (0.28 ml, 2.4 mmol),triethylamine (0.28 ml, 2 mmol), palladium acetate (15 mg, 0.07 mmol)and tri-o-tolylphosphine (40 mg, 0.14 mmol) in acetonitrile (20 ml) washeated under reflux for 16 hours and then evaporated under reducedpressure. The residue was partitioned between dichloromethane (50 ml)and saturated aqueous sodium carbonate solution (50 ml), then theorganic phase separated, dried (MgSO₄) and evaporated under reducedpressure. The crude product was crystallised from ether to provide thetitle compound (145 mg), m.p. 157-158° C. Found: C,66.24; H,4.87;N,12.20. C₂₅H₂₁F₃N₄O requires C,66.66; H,4.70; N,12.44%.

EXAMPLE 3Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[4-(2-methylsulphonylethenyl)phenyl]butan-2-ol

Obtained from(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4-iodophenyl)butan-2-ol(Preparation 11) and methyl vinyl sulphone by a procedure similar tothat described in Example 2. M.p. 169-171° C. Found: C,58.70; H,5.00;N,9.29. C₂₁H₂₁F₂N₃O₃S; 0.25 (C₂H₅)₂O requires C,58.46; H,5.24; N,9.30%.

EXAMPLE 4Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[4-(2-carbamoylethenyl)phenyl]butan-2-ol

Obtained from the title compound of Preparation 11 and acrylamide by aprocedure similar to that described in Example 2. M.p. 122-124° C.Found: C,61.82; H,5.55; N,12.53. C₂₁H₂₀F₂N₄O₂; 0.50 H₂O; 0.33 CH₃CO₂C₂H₅requires C,61.42; H,5.46; N,12.84%.

EXAMPLE 5Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[2-(imidazol-1-yl)ethenyl]phenyl}butan-2-ol

Obtained from the title compound of Preparation 11 and 1-vinylimidazoleby a procedure similar to that described in Example 2. M.p. 225-227° C.Found: C,65.34; H,5.24; N,16.39. C₂₃H₂₁F₂N₅O requires C,65.55; H,5.02;N,16.62%.

EXAMPLE 6Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-[2-(pyrid-2-yl)ethenyl]pyrid-2-yl}butan-2-ol

Obtained from the title compound of Preparation 3 and 2-vinylpyridine bya procedure similar to that described in Example 2. M.p. 139-141° C.Found: C,66.51; H,4.94; N,15.98. C₂₄H₂₁F₂N₅O requires C,66.50; H,4.88;N,16.16%.

EXAMPLE 7Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-(2-carbamoylethenyl)pyrid-2-yl}butan-2-ol

Obtained from the title compound of Preparation 3 and acrylamide by aprocedure similar to that described in Example 2. M.p. 170-172° C.Found: C,59.74; H,4.63; N,17.91. C₂₀H₁₉F₂N₅O₂ requires C,60.14; H,4.79;N,17.54%.

EXAMPLE 8Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-[2-(1-ethoxymethyl-1,2,3-triazol-5-yl)ethenyl]pyrid-2-yl}butan-2-ol

Obtained from the title compound of Preparation 3 and1-ethoxymethyl-5-vinyl-1,2,3-triazole (Preparation 15) by a proceduresimilar to that described in Example 2 except that 0.3 mol. equiv. ofpalladium acetate was used and also, instead of tri-o-tolylphosphine,1,1′-bis(diphenylphosphino)ferrocene (0.3 mol. equiv.). M.p. 161-163° C.Found: C,59.73; H,5.31; N,19.77. C₂₄H₂₅F₂N₇O₂; 0.10 CH₃CO₂C₂H₅ requiresC,59.77; H,5.30; N,20.00%.

EXAMPLE 9Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-[5-{3-hydroxy-3-methylbut-1-en-1-yl}pyrid-2-yl]butan-2-ol

Obtained from the title compound of Preparation 3 and2-methylbut-3-en-2-ol by a procedure similar to that described inExample 2. M.p. 159-161° C. Found: C,63.67; H,6.22; N,13.16.C₂₂H₂₄F₂N₄O₂ requires C,63.76; H,5.84; N,13.52%.

EXAMPLE 10Trans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-[2-(1,2,3-triazol-4-yl)ethenyl]pyrid-2-yl}butan-2-ol

A stirred solution of(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{5-[2-(1-ethoxymethyl-1,2,3-triazol-5-yl)ethenyl]pyrid-2-yl}butan-2-ol(Example 8; 0.1 g, 0.21 mmol) in a mixture of hydrochloric acid (2M; 2ml), water (2 ml) and ethanol (4 ml) was heated under reflux for 30minutes. The bulk of the ethanol was evaporated under reduced pressure,the concentrated reaction solution basified with saturated aqueoussodium carbonate solution and extracted with dichloromethane (4×30 ml),then the combined extracts dried (Na₂SO₄) and evaporated under reducedpressure to furnish a brown oil. Trituration of the oil with ether gavean off-white solid which, on crystallisation from hexane:ethyl acetate,afforded the title compound (30 mg), m.p. 192-193° C. Found: C,59.44;H,4.54; N,22.93. C₂₁H₁₉F₂N₇O requires C,59.57; H,4.52; N,23.16%.

Preparation 1 2-Ethyl-5-bromopyridine

A solution of ethylmagnesium bromide in dry ether (3M; 100 ml, 0.30 mol)was added dropwise to a stirred, ice-cooled solution of anhydrous zincchloride (40.9 g, 0.30 mol) in dry tetrahydrofuran (500 ml) undernitrogen and the resulting solution stirred for a further 1 hour beforethe sequential addition of tetrakis (triphenylphosphine)palladium(0)(1.0 g, 0.87 mmol) and a solution of 2,5-dibromopyridine (50 g, 0.21mol) in dry tetrahydrofuran (200 ml). The resulting yellow suspensionwas stirred at room temperature for 18 hours, quenched by the additionof water (200 ml) and evaporated under reduced pressure. The residue waspartitioned between dichloromethane (500 ml) and a suspension ofethylenediaminetetraacetic acid (200 g) in water (1 l). The organicphase was separated, combined with a dichloromethane extract (500 ml) ofthe aqueous phase, dried (MgSO₄) and evaporated under reduced pressure.Distillation under reduced pressure of the residue gave the titlecompound (28.8 g) as a colourless oil, b.p. 123-124° C./8 kPa (60 mmHg). δ(CDCl₃): 1.30(3H,t), 2.80(2H,q), 7.10(1H,d), 7.70(1H,dd),8.60(1H,d).

Preparation 2 2-(1-Bromoethyl)-5-bromopyridine

A stirred solution of the title compound of Preparation 1 (1.86 g, 10mmol) and N-bromosuccinimide (1.78 g, 10 mmol) in 1,2-dichloroethane (20ml) was heated to reflux, α,α′-azobis(isobutyronitrile) (20 mg) addedand the reaction solution stirred under reflux for 2 hours. Theresulting, cool suspension was filtered, the filtrate evaporated underreduced pressure and the residue purified by column chromatography onsilica gel, using hexane:dichloromethane (1:1) as eluant, to provide thetitle compound (2.12 g) as a pale yellow oil. δ(CDCl₃): 2.05(3H,d),5.20(1H,q), 7.35(1H,d), 7.80(1H,d), 8.60(1H,d).

Preparation 3(2R,3S/2S,3R)-1-(1,2,4-Triazol-1-yl)-2-(2,4-difluorophenyl)-3-(5-bromopyrid-2-yl)butan-2-ol

A solution of the title compound of Preparation 2 (1.32 g, 5 mmol) and1-(2,4-difluorophenacyl)-1,2,4-triazole (1.11 g, 5 mmol) in drytetrahydrofuran (12 ml) was added dropwise to a stirred suspension ofzinc powder (0.85 g, 13 mmol) in dry tetrahydrofuran (8 ml) at roomtemperature under nitrogen. Subsequent addition of iodine (0.25, 1 mmol)caused an exothermic reaction, after which the reaction mixture wasquenched by the sequential addition of glacial acetic acid (1 ml) andwater (10 ml), then ethyl acetate (30 ml) and ethylenediaminetetraaceticacid disodium salt dihydrate (3.72 g, 10 mmol) were added. The organicphase was separated, dried (MgSO₄) and evaporated under reducedpressure, then the residue purified by column chromatography on silicagel using hexane:ethyl acetate (1:1) as eluant, followed by triturationwith ether, to furnish the title compound (0.62 g), m.p. 158-161° C.δ(CDCl₃): 1.08(3H,d), 4.05 and 4.78(2H,AB system). Found: C,49.81;H,3.55; N,13.45. C₁₇H₁₅BrF₂N₄O requires C,49.90; H,3.69; N,13.69%.

Further elution of the above column using hexane:ethyl acetate (1:2)afforded the undesired, minor (2R,3R/2S,3S) diastereoisomeric pair ofenantiomers as an oil (0.22 g), which crystallised (m.p. 82-83° C.) onstanding at room temperature. δ(CDCl₃): 1.50(3H,d), 4.66 and 4.80(2H, ABsystem). Found: C,49.96; H,3.54; N,13.70. C₁₇H₁₅BrF₂N₄O requiresC,49.90; H,3.69; N,13.69%.

Preparation 4 N,O-Dimethyl-4-iodobenzenehydroxamic acid

A solution of pyridine (104 g, 1.32 mol) in dichloromethane (150 ml) wasadded dropwise to an ice-cooled, stirred suspension of 4-iodobenzoylchloride (251 g, 0.94 mol) and N,O-dimethylhydroxylamine hydrochloride(97 g, 0.94 mol) in dichloromethane (850 ml). The mixture was allowed towarm to room temperature and then stirred for a further 18 hours. Theresulting solution was evaporated under reduced pressure, the residuedissolved in ethyl acetate (1 l) and this solution then washedsequentially with hydrochloric acid (2M, 3×400 ml) and saturated aqueoussodium bicarbonate solution (300 ml), dried (Na₂SO₄) and evaporatedunder reduced pressure. The residue was purified by distillation underreduced pressure to give the title compound (241 g) as a yellow oil,b.p. 130° C./13.3 Pa (0.1 mm Hg). δ(CDCl₃): 3.32(3H,s), 3.50(3H,s),7.40(2H,d) 7.72(2H,d).

Preparation 5 1-(4-Iodophenyl)-2-(2,4-difluorophenyl)ethanone

2,4-Difluorobenzyl bromide (23.7 ml, 0.114 mol) was added dropwise to astirred mixture of magnesium turnings (8.1 g, 0.183 mol) in dry ether(300 ml) under nitrogen. The mixture was warmed initially untilinitiation of the reaction occurred and, thereafter, said bromide wasadded at such a rate as to maintain gentle reflux. After 1 hour, theresulting solution of the Grignard reagent was added dropwise to asolution of the title compound of Preparation 4 (45.71 g, 0.157 moo indry ether (300 ml) at about −70° C. and the mixture allowed to warm toroom temperature overnight (18 hours). The resulting mixture waspartitioned between saturated aqueous ammonium chloride solution andethyl acetate, then the organic phase separated, dried (MgSO₄) andevaporated under reduced pressure, to provide the title compound (38.71g) as a white solid. δ(CDCl₃): 4.23(2H,s), 6.83(2H,m), 7.17(1H,dt,J=7.0and 8.5 Hz), 7.72(2H,d,J=9.0 Hz), 7.84(2H,d,J=9.0 Hz).

Preparation 6 1-(4-Iodophenyl)-2-(2,4-difluorophenyl)prop-2-enone

Bis(dimethylamino)methane (8.78 ml, 0.075 mol) was added dropwise to astirred suspension of the title compound of Preparation 5 (17.73 g,0.0495 mol) in acetic anhydride (23.1 ml, 0.248 mol) at roomtemperature. There was an exothermic reaction, the temperature of themixture rising to about 60° C. After the end of the addition the mixturewas stirred at room temperature for 35 minutes, then ice-water added tohydrolyse the excess acetic anhydride. After a further 30 minutes, theorganic material was extracted into ethyl acetate and the combinedextracts washed sequentially with dilute hydrochloric acid and saturatedaqueous sodium bicarbonate solution, dried (MgSO₄) and evaporated underreduced pressure to furnish the title compound (17.03 g) as a whitesolid. δ(CDCl₃): 5.90(1H,s), 6.14(1H,s), 6.84(1H,ddd,J=2,8 and 12 Hz),6.95(1H,dt,J=2 and 8 Hz, 7.39(1H,dt,J=7 and 9 Hz), 7.59(2H,d,J=9 Hz),7.83(2H,d,J=9 Hz).

Preparation 7 (2R,2S)-2-(2,4-Difluorophenyl)-2-(4-iodobenzoyl)oxirane

Benzyltrimethylammonium hydroxide (40% aqueous solution; 3.44 ml, 8.2mmol) was added in one portion to a solution of the title compound ofPreparation 6 (37.3 g, 100.8 mmol) and t-butylhydroperoxide (3M intrimethylpentane; 36.6 ml, 109 mmol) in toluene (550 ml) at roomtemperature. After 2 hours, the mixture was wasbed with water (2×500ml), dried (MgSO₄) and evaporated under reduced pressure to afford thetitle compound (37.46 g) as a white solid. δ(CDCl₃): 3.22(1H,d,J=5 Hz),3.42(1H,d,J=5 Hz), 6.80(1H,ddd,J=2,8 and 12 Hz), 6.93(1H,dt,J=2 and 8Hz), 7.47(1H,dt,J=7 and 9 Hz), 7.70(2H,d,J=9 Hz), 7.77(2H, d, J=9 Hz).

Preparation 8(2R,2S)-2-(2,4-Difluorophenyl)-2-[1-(4-iodophenyl)ethenyl]oxirane

n-Butyllithium (2.5M in hexane; 50 ml, 125 mmol) was added dropwise over10 minutes to a stirred suspension of methyltriphenylphosphonium bromide(45.0 g, 126 mmol) in dry tetrahydrofuran (600 ml) at about −70° C.under nitrogen. The mixture was allowed to warm to −20° C. over 20minutes, then a solution of the title compound of Preparation 7 (37.46g, 97 mmol) in dry tetrahydrofuran (200 ml) was added over 5 minutes.This mixture was allowed to warm to room temperature, stirred for afurther 84 hours, treated with 10% aqueous ammonium chloride solution(500 ml) and concentrated under reduced pressure. The organic materialwas extracted into ethyl acetate and the combined extracts dried (MgSO₄)and evaporated under reduced pressure. The resulting solid residue wastreated with boiling hexane (3×500 ml) and the remaining soliddiscarded. The combined hexane solutions were filtered through a shortpad of silica gel and evaporated under reduced pressure to give thetitle compound (34.3 g) as a yellow oil. δ(CDCl₃): 3.13(1H,d,J=5 Hz),3.17(1H,d,J=5 Hz), 5.45(2H,m), 6.72(1H,m), 6.80(1H,m), 7.14(2H,d,J=9Hz), 7.39(1H,dt,J=7 and 9 Hz), 7.60(2H,d,J=9 Hz).

Preparation 9(2R,2S)-1-(1,2,4-Triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4-iodophenyl)but-3-en-2-ol

Sodium 1,2,4-triazole (12.15 g, 133 mmol) was added to a solution of thetitle compound of Preparation 8 (34.3 g, 89 mmol) in drydimethylformamide (350 ml) at about 70° C. under nitrogen. The resultingmixture was stirred for 5 hours, then allowed to cool and the solventremoved by evaporation under reduced pressure. The residue was dissolvedin ether (800 ml) and the solution washed with water (2×500 ml), dred(MgSO₄) and treated with silica gel (60-200μ; 75 g). The ether wasevaporated under reduced pressure and the residual solid applied to asilica gel column (40-60μ; 300 g). Elution with a solvent gradient ofhexane:ethyl acetate (100:0 to 25:75), followed by evaporation underreduced pressure of the required eluate fractions, provided the titlecompound (23.8 g) as a white foam. δ(CDCl₃): 4.55(1H,d,J=15 Hz),4.90(1H,d,J=15 Hz), 5.16(1H,s), 5.25(2H,s), 6.70(2H,m), 7.03(2H,d,J=9Hz), 7.43(1H,dt,J=7 and 9 Hz), 7.58(2H,d,J=9 Hz), 7.79(1H,s),7.80(1H,s).

Preparation 10 (2R)- and(2S)-1-(1,2,4-Triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4-iodophenyl)but-3-en-2-ol

The title compound of Preparation 9 was resolved by chiral HPLC using a“Chiralpak AD” (Trade Mark) column and hexane:ethanol (95:5) as eluant.Fractions containing each enantiomer were combined and evaporated underreduced pressure, then the residues further purified by columnchromatography on silica gel, using dichloromethane:methanol (95:5) aseluant, followed by trituration with ether.

Analytical HPLC indicated >99% ee for each enantiomer:

for (A), the required (2R)-enantiomer (peak 2), m.p. 111-112° C. and[α]_(D) ²⁵−49° (c=0.1, CH₃OH). Found: C,47.52; H,2.97; N,9.09.C₁₈H₁₄F₂IN₃O requires C,47.70; H,3.11; N,9.27%.

for (B), the (2S)-enantiomer (peak 1), m.p 110-111° C. and [α]_(D)²⁵+41° (c=0.1, CH₃OH). Found: C,47.88; H,3.02; N,9.29. C₁₈H₁₄F₂IN₃Orequires C,47.70; H,3.11; N,9.27%.

Preparation 11(2R,3S/2S,3R)-1-(1,2,4-Triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4-iodophenyl)butan-2-ol

A stirred solution of the title compound of Preparation 9 (1.0 g, 2.2mmol) and p-toluenesulphonylhydrazide (4.1g, 22 mmol) in toluene (30 ml)was heated under reflux for 3 hours, allowed to cool to roomtemperature, diluted with ethyl acetate (30 ml) and washed with aqueoussodium hydroxide solution (2M; 2×30 ml). The combined aqueous washingswere washed with ethyl acetate, then the combined organic solutionswashed with saturated brine (30 ml), dried (MgSO₄) and evaporated underreduced pressure. The residue was purified by column chromatography onsilica gel, using an elution gradient of hexane:ethyl acetate (3:1 to1:3), whereupon the first diastereoisomeric pair of enantiomers to elutewas the title compound (0.30 g), obtained as a white solid, m.p.169-171°, after trituration with hexane containing a trace of ethylacetate. δ(CDCl₃): 1.10(3H,d), 3.30(1H,q), 3.80 and 4.80(2H,AB system),4.90(1H,s), 6.80(2H,m), 7.30(2H,m), 7.45(1H,m), 7.70(4H,m). Found:C,47.66; H,3.55; N,9.19. C₂₂H₂₅F₃ClN₅O₃ requires C,47.49; H,3.54;N,9.23%.

Preparation 12(2R,3S)-1-(1,2,4-Triazol-1-yl)-2-(2,4-difluorophenyl)-3-(4iodophenol)butan-2-ol

Obtained from the title compound of Preparation 10A by a proceduresimilar to that described in Preparation 11 followed by crystallisationfrom aqueous ethanol, m.p. 104° C. and [α]_(D) ²⁵ −44° (c=0.1, CH₃OH).Found: C,47.42; H,3.64; N,9.11. C₂₂H₂₅F₃ClN₅O₃ requires C,47.49; H,3.54;N,9.23%. m/z 456(M+1)⁺.

Preparation 13 1-Ethoxymethyl-1,2,3-triazole

Chloromethyl ethyl ether (125 g, 1.32 mole) was added dropwise over 1.5hours to an ice-cooled, stirred suspension of 1,2,3-triazole (91.4 g,1.32 mole) and anhydrous potassium carbonate (183 g, 1.32 mole) inacetone (1.5 l). The mixture was then allowed to warm to roomtemperature and stirred for a further 18 hours. The solvent wasevaporated under reduced pressure, the residue dissolved in water (1 l)and the aqueous solution extracted with dichloromethane (3×300 ml). Thecombined extracts were washed with saturated brine (3×300 ml), dried(Na₂SO₄) and evaporated under reduced pressure to furnish a pale yellowoil.

Distillation under reduced pressure of the oil initially afforded2-ethoxymethyl-1,2,3-triazole (57 g), b.p. <90°C./0.4 kPa (3 mm Hg).δ(CDCl₃): 1.17(3H,t), 3.60(2H,q), 5.70(2H,s), 7.70(2H,s). Found:C,47.36; H,7.23; N,32.62. C₅H₉N₃O requires C,47.19; H,7.14; N,33.05%.

Continued distillation yielded the title compound (73 g), b.p. 92-93°C./0.4 kPa (3 mm Hg). δ(CDCl₃): 1.15(3H,t), 3.56(2H,q), 5.70(2H,s),7.77(1H,s), 7.79(1H,s). Found: C,46.30; H,7.52; N,33.29. C₅H₉N₃Orequires C,47.19; H,7.14; N,33.05%.

Preparation 14 1-Ethoxymethyl-5-formyl-1,2,3-triazole

n-Butyllithium (2.5M in hexane; 11.3 ml, 28.3 mmol) was added dropwiseto a stirred solution of the title compound of Preparation 13 (3.0 g,23.6 mmol) in dry tetrahydrofuran (100 ml) at about −70° C. undernitrogen. The white suspension was stirred for a further 30 minutes,then a solution of dry dimethylformamide (3.66 ml, 47.2 mmol) in drytetrahydrofuran (3 ml) was added dropwise. The resulting solution wasallowed to warm to room temperature, stirred for a further 30 minutesand treated with saturated aqueous ammonium chloride solution (20 ml)and water (30 ml). The organic phase was separated, combined withdichloromethane extracts (2×50 ml) of the aqueous phase, dried (Na₂SO₄)and evaporated under reduced pressure. The crude product was purified bycolumn chromatography on silica gel, using an elution gradient ofhexane:ethyl acetate (3:1 to 2:1), to afford the title compound (1.0 g)as a colourless oil. δ(CDCl₃): 1.15(3H,t), 3.60(2H,q), 6.00(2H,s),8.25(1H,s), 10.10(1H,s). m/z 156 (M+1)⁺.

Preparation 15 1-Ethoxymethyl-5-vinyl-1,2,3-triazole

Obtained from the title compound of Preparation 14, by a proceduresimilar to that described in Preparation 8, as a colourless oil.δ(CDCl₃): 1.15(3H,t), 3.50(2H,q), 5.55(1H,d), 5.70(2H,s), 5.90(1H,d),6.70(1H,dd), 7.80(1H,s).

Preparation 16 1-Methyl-5-formylpyrazole

Obtained from 1-methylpyrazole by a procedure similar to that describedin Preparation 14. δ(CDCl₃): 4.20(3H,s), 6.90(1H,s), 7.55(1H,s),9.85(1H,s).

Preparation 17 1-Methyl-5-vinylpyrazole

Obtained from the title compound of Preparation 16 by a proceduresimilar to that described in Preparation 8. δ(CDCl₃): 3.90(3H,s),5.35(1H,d), 5.70(1H,d), 6.40(1H,d), 6.60(1H,dd), 7.40(1H,d).

Biological Activity

The Table below illustrates the in vivo activity against acute systemiccandidosis in immune-normal mice for a selection of the compounds of theinvention.

Mice were infected intravenously with Candida albicans in order toestablish a systemic infection (all untreated control mice died by 2days post-infection). Compound efficacy was assessed on the basis ofsurvival after oral dosing (0.1-5 mg/kg; 1, 4 and 24 hourspost-infection) and was measured as the dose protecting 50% of mice onday 2 post-infection.

TABLE EXAMPLE PD₅₀ (mg/kg) 1 <0.10 5 0.18 9 0.18 10 0.32

Safety Profile

The compounds of the invention have not been found to exhibit any overtsigns of toxicity. For example, in a 7-day toxicity study in rats (30mg/kg p.o., o.d.); Example 1 elicited no adverse effects.

What is claimed is:
 1. A compound of formula (I)

or a pharmaceutically acceptable salt thereof, wherein X is CH; R¹ isphenyl substituted with 1 to 3 substituents each independently selectedfrom halo and CF₃; R² is Het; and Het is imidazolyl, wherein theforegoing Het groups are optionally substituted with C₁-C₄ alkyl or(C₁-C₄ alkoxy)methyl.
 2. A compound according to claim 1, wherein R¹ isphenyl substituted by 1 or 2 substituents each independently selectedfrom F and Cl; and R² is Het.
 3. A compound according to claim 2 whereinR¹ is 2,4-difluorophenyl; and R² is imidazol-1-yl.
 4. A compoundaccording to claim 1 which is the (2R,3S)-enantiomer of formula (1A):


5. A compound according to claim 1 which is the trans-alkene isomer. 6.The compound according to claim 5 which istrans-(2R,3S/2S,3R)-1-(1,2,4-triazol-1-yl)-2-(2,4-difluorophenyl)-3-{4-[2(imidazole-1-yl)ethenyl]phenyl}butan-2-olor a pharmaceutically acceptable salt thereof.
 7. A pharmaceuticalcomposition comprising a compound of claim 1, together with apharmaceutically acceptable diluent or carrier.
 8. A method of treatinga fungal infection in a mammal which comprises treating said mammal withan effective amount of a compound of claim 1.